The invention relates to an analysis card making it possible to count and characterize microorganisms or any other biological material, such as a nucleic acid or antibody, in a biological sample. The invention also relates to a method of filling such a card as well as to the use of this card.
The terms [sic] xe2x80x9cbiological samplexe2x80x9d should be understood to mean a liquid sample resulting from the physical treatment, for example homogenization, or dissolution in a solvent, or from the biological treatment, such as enzymatic digestion, of solid or liquid products, possibly consisting of food products, of water, of blood.
Apart from the conventional counting method in Petri dishes, nonautomated methods are found in the prior art for quantifying microorganisms; these methods are mainly based on determining the Most probable number (MPN).
U.S. Pat. No. 5,700,655 describes an apparatus for quantification of biological material in a liquid sample. This apparatus consists of a plastic sheet obtained by thermoforming, the sheet itself consisting of analysis cavities of the same size and of a cover making it possible to maintain sterility. The method consists in firstly depositing the sample to be analyzed in the center of the sheet and then the reaction mixture supplied with the apparatus. The apparatus thus prepared is agitated so as to fill all the cavities in the identical manner and then the excess reaction mixture is eliminated. Finally, the sheet is incubated for a time long enough to reveal the presence of microbes in one or more wells.
However, this apparatus, like the conventional Petri dishes, only allows the sample to be diluted once. Knowing that the sample has to be diluted several times in order to be sure of being able to determine the concentration of germs contained in said sample, it will be necessary to repeat this operation at least once.
In addition, this apparatus has another major drawback. This drawback consists of the need to eliminate the surplus reaction mixture. Since this operation is carried out by tilting the sheet, certain cavities may lose a part of their content. The amount of liquid in the cavities will therefore no longer be identical, and consequently the determination of the amount of germs will be false.
Furthermore, eliminating the surplus reaction mixture results in the production of potentially contaminated waste, which has to be reprocessed. Likewise, this elimination of the surplus contaminates the outside of the apparatus, which then becomes a source of biological contamination for the operator and all other surfaces which come into contact with him.
Finally [sic], since the cavities are arranged concentrically, the read-out also takes place concentrically, which may be a source of error, especially as the positive cavities are often dispersed over the sheet.
Finally, the abovementioned apparatus still has the basic drawback of Petri dishes, namely the fact that it must absolutely be handled with care and horizontally while the medium is gelling, if this gelling function is provided, so as to prevent a sample from leaking or passing from one cavity to another, which results in handling artifacts.
U.S. Pat. No. 5,518,892 discloses another apparatus for quantification of microorganisms in a liquid medium. It comprises a bag, made in a material and obtained by thermoforming, which has an opening on one of its sides. The method consists in forming, by heating, impermeable compartments or cavities on the lower face of the bag, allowing various amounts of the sample to be separated. Once the wells have been formed, the reaction mixture is prepared by adding the sample to be analyzed to the reaction mixture supplied with the apparatus. The reaction mixture is then poured into the bag via the opening provided for this purpose. A check is made that all the cavities are properly filled and then, after heat sealing, the bag is incubated for a time long enough to reveal the presence of microorganisms in one or more cavities. The cavities may be of the same size or three series of different sizes. It is thus possible to use a single bag in order to simulate seeding according to several dilutions.
However, this system has certain drawbacks. Firstly, and unlike an automated and very precise industrial injection molding system, this apparatus cannot guarantee optimum molding quality making it possible to obtain cavities of perfectly identical capacity, since basically simple plastic sheets are used which are formed by thermoforming. This is particularly important when a molding is produced with series of cavities of various volumes, especially when these are small volumes. Secondly, the reaction mixture is introduced by hand and the distribution in the cavities therefore takes place randomly. Thus, there may be no certainty that the volume of reaction mixture delivered into each cavity is the same, even if the size of the cavities is perfectly identical. The results may therefore again be false. Thirdly, the cavities are isolated by heat sealing at temperatures above 180xc2x0 C., which carries the risk of irreversibly impairing (destroying the biological material to be analyzed and/or evaporating all or some of the liquid containing said material) the sample fraction contained in the cavities. This is all the more sensitive the smaller the size of the cavities and the smaller the amount of liquid they contain. This handling may therefore falsify the results of the counting and of the characterization.
According to the present invention, the analysis card proposed helps to overcome all of the abovementioned drawbacks.
This card makes it possible to fill all the cavities, whatever their size, which is proportional from one cavity to another, and which does not require positioning precautions to be taken when filling it. By xe2x80x9cproportional from one cavity to anotherxe2x80x9d is meant that when two cavities have the same dimensions, the proportion will then be substantially 1 for 1, or when two cavities have different dimensions, the proportion is different from 1 for 1. Moreover, there is no risk of contamination, on the one hand, of the specimen with the outside, since the liquid remains confined, and, on the other hand, between the various cavities of the card, since an isolating separation is also provided between each cavity of said card.
For this purpose, the present invention relates, according to a first preferred embodiment, to an analysis card which comprises a body consisting of:
a region for injecting a biological liquid to be analyzed into the card,
a main feed channel for the biological liquid coming from the injection region,
at least two secondary channels lying in the extension of the main channel, and
at least one well corresponding to each secondary channel,
characterized in that each well constitutes a means of delivering a predetermined volume of said biological liquid into at least two terminal analysis cavities, via a terminal channel for each analysis cavity, and in that the total volume to be analyzed is less than the total volume of all of the analysis cavities.
According to a second preferred embodiment of the invention, the analysis card which comprises a body consisting of:
a region for injecting a biological liquid to be analyzed into the card,
at least two feed channels for the biological liquid coming from the injection region, and
at least one well corresponding to each feed channel,
[lacuna] characterized in that each well constitutes a means of delivering a predetermined volume of said biological liquid into at least two terminal analysis cavities, via a terminal channel for each analysis cavity, and in that the volume to be analyzed is less than the total volume of all of the analysis cavities.
According to the above two embodiments, the delivery of the biological liquid into the terminal analysis cavities takes place in parallel.
According to a third preferred embodiment of the invention, the analysis card which comprises a body consisting of:
a region for injecting a biological liquid to be analyzed into the card,
a main feed channel for the biological liquid coming from the injection region,
at least two secondary channels lying in the extension of the main channel, and
at least one cavity corresponding to each secondary channel,
characterized in that each cavity constitutes a means of receiving a predetermined volume of said biological liquid and in that the volume to be analyzed, introduced into the card, is greater than the total volume of all of the analysis cavities.
According to the previous embodiment, the delivery of the biological liquid into the analysis cavities takes place in series and is followed by a purge. According to the first embodiment, a well, acting as a main means of delivering the biological liquid, is present between, on the one hand, the main channel and, on the other hand, the secondary channels.
According to one of the two embodiments, the volumes of the secondary channels are all identical.
In a preferred version of one of these three embodiments, the terminal analysis cavities are of at least two different volumes.
Preferably, the terminal analysis cavities are of three different volumes.
The ratio existing between two cavities of different volumes is between 1/5 and 1/20, 1/50 and 1/200 or 1/500 and 1/2000 and especially 1/10, 1/100 or 1/1000.
The cavities of the same volume are arranged geometrically, for example aligned or concentric, in order to facilitate the manual and/or automatic optical read-out.
The present invention also relates to the method of filling an analysis card, as presented in the first two embodiments, characterized in that it consists in:
connecting the injection region of the card to a volume of the biological sample to be analyzed,
creating a vacuum within and/or in the vicinity of said card,
breaking the vacuum so as, on the one hand, to transfer the biological liquid into the analysis cavities and, on the other hand, to isolate with an inert fluid the various fractions of the sample which come from said biological liquid and which are present in each of said analysis cavities, and
analyzing the result obtained so as to determine the amount of microorganisms or of any other biological material, such as nucleic acids, antibodies, present in the initial biological sample.
The present invention also relates to the method of filling an analysis card as presented in the third embodiment, characterized in that it consists in:
connecting the injection region or the outlet of the card to a volume of a biological sample to be analyzed,
creating a vacuum or an overpressure within and/or in the vicinity of said card so as to transfer the biological liquid into the cavities,
stopping the vacuum or the overpressure,
purging the main channel in order to isolate with an inert fluid the various fractions of the sample which come from said biological liquid and which are present in each of said analysis cavities, and
analyzing the result obtained so as to determine the amount of microorganisms or of any other biological component, such as nucleic acids, antibodies, present in the initial biological sample.
According to the first two embodiments of the card, once the vacuum has been broken, the isolating fluid is in contact with the biological liquid either in the secondary channels or feed channels or in the analysis cavities.
Preferably, the volume of the biological liquid to be analyzed is less than the total volume of all of the analysis cavities.
According to the third embodiment of the card, the volume of the biological liquid to be analyzed is greater than or equal to the total volume of all of the analysis cavities, of all of the fluid circuit and of the overflow.
Preferably, the volume of the biological liquid to be analyzed is greater than or equal to the total volume of all of the analysis cavities and of all of the fluid circuit.
According to the first two embodiments of the card, the ratio of the volume of the biological sample to be analyzed to the total volume of all of the analysis cavities is, for example, from 1/10 to 9/10, especially from 4/10 to 6/10, and preferably 5/10 .
Finally, the present invention relates to the use of an analysis card, as described above, for counting microorganisms using the technique of the most probable number.